The Impact Of High Serum Progesterone Concentration On The Day Of And The Day After HCG Administration On The Endometrium In Peri-implantation Period In Women Undergoing IVF Treatment

PART ONE:A comparison of progesterone measurement on day of hCG administration with measurement on the day after hCG administration in IVF-ET cyclesAim:Elevated progesterone on the day of human chorionic gonadotropin (hCG) administration was associated with adverse pregnancy outcomes in women undergoing IVF-ET treatment. However, it is not known if progesterone measurement on the day after hCG administration is more informative than progesterone measurement on the day of hCG administration. The aim of this study is to investigate if progesterone measurement on the day after hCG administration has any additional clinical value, over and about what can be obtained from progesterone measurement on the day of hCG administration? Methods:This was a single-center, non-interventional retrospective, observational, cohort of a consecutive series of5828ovarian stimulation IVF cycles in the Reproductive Center, Sir Run Run Shaw Hospital, Medical School of Zhe Jiang University between January2011and May2013.All subjects included in the study were advised not to proceed with fresh embryo transfer in the treatment cycle but to have embryos frozen for replacement in a subsequent cycle, according to our establish clinical protocol to avoid ovarian hyperstimulation syndrome, either because there were more than15oocytes retrieved or because the progesterone level on the day of hCG administration was elevated to≥1.5ng/ml.During the period of study, it was our routine practice to obtain blood sample for progesterone measurement on the day of hCG administration and the day after hCG administration. All blood samples obtained consistently in the morning, and the same hormone assay was used throughout the study. There was no change in laboratory protocol, and the implantation rate and pregnancy rate were stable.To investigate the association of the progesterone measurements (progesterone level on the day of hCG administration and progesterone level on the day after hCG administration) with embryo implantation rate, overall1457cycles with fresh embryo transfer were included for further analysis.To determine the relative contribution of the progesterone measurement on the day of hCG administration and the day after hCG administration on the implantation rate, multivariable stepwise regression analysis was performed by using implantation rate as the dependent variable, and following factors as independent variables:age, the number of oocytes retrieved, endometrium thickness on the day of hCG administration, E2level on the day of hCG administration, P level on the day of hCG administration, E2level on the day after hCG administration, P level on the day after hCG administration, sum of P level on the day of hCG administration and P level on the day after hCG administration, total rFSH dose used, duration of rFSH stimulation, the number of embryos transferred per ET, and the number of high-quality embryo transferred per ET.According to a consecutive series of5828samples routinely collected from patients undergoing ovarian stimulation treatments for IVF from2011-2013in our hospital, the90%percentile was used as the cut-off to define high progesterone level, which was1.7ng/ml on the day of hCG administration and9.5ng/ml on the day after hCG administration.According to the serial progesterone concentrations on the day of hCG administration and on the day after hCG administration, patients were categorized into4groups:group NN, progesterone level normal on the day of hCG and day after hCG administration; group NH, progesterone level normal on the day of hCG but high on the day after hCG administration; group HN, progesterone level high on the day of hCG but normal on the day after hCG administration; group HH, progesterone level high on the day of hCG and day after hCG administration. The clinical features and outcome of treatment of the4subgroups of subjects were further analyzed Results:1. According to the progesterone level on the day of hCG administration and the progesterone level on the day after hCG administration of a consecutive5828IVF ovarian stimulated cycles, the mean±standard deviation of progesterone concentration on the day of hCG administration was1.4±0.8ng/ml, which was increased significantly (p<0.001) to7.9±5.5ng/ml on the day after hCG administration.2. Progesterone rises rapidly after hCG administration, and increased by about5folds over a24hour period.3. Based on the measurements of progesterone on the day of hCG administration and on the day after hCG administration from a consecutive5828IVF ovarian stimulated cycles, the cut-off value of progesterone on the day of hCG administration was1.7ng/ml; the cut-off value of progesterone on the day after hCG administration was9.5ng/ml.4. The regression model selected the following variables in decreasing order of importance:the number of high quality embryos transferred per ET, the age of subjects, and sum of P level on the day of hCG administration and P level on the day after hCG administration. Had the sum of P level on the day of hCG administration and P level on the day after hCG administration been removed as an independent variable, P level on the day after hCG administration would be selected instead as the third variable in the model. In addition, if P level on the day after hCG administration was also removed from the list of independent variables, P level on the day of hCG administration would be selected as the third variable instead.5. Overall1457fresh ET cycles were categorized into4groups according to whether or not the progesterone on the day of hCG and progesterone on the day after hCG were high or normal. The results showed that:compared to women with normal progesterone measurements on both days, women with high progesterone measurements on both days (day of hCG and day after hCG administration) appeared to be younger (31.9±4.6years&29.7±3.5years, P<0.001), with a lower basal FSH result (8.1±1.9IU/L&7.9±1.2IU/L, P<0.05), had a higher number of oocytes retrieved (7.9±4.0&12.7±3.3, P<0.001), produced a higher number of embryos (5.3±3.0&6.7±2.6, P<0.001), although the number of embryos transferred was not significant different (1.8±0.4&1.8±0.4), the number of good embryos transferred was not significant different (1.6±0.5&1.7±0.5),but achieved lower implantation (36.0%&22.2%, P<0.001) and clinical pregnancy rates (59.7%&37.5%, P<0.001).Conclusion:Progesterone measurement should be considered not only on the day of hCG administration but also the day after hCG administration to provide clinically useful information on whether or not embryos should be frozen and transfer deferred to a subsequent cycle. PAER TWO:The impact of high progesterone level prior to oocyte retrieval on endometrial morphology and uNK cell count in the peri-implantation periodAim:High progesterone level prior to oocyte retrieval has been reported to adversely affect endometrial receptivity, resulting in reduced implantation rate. The exact molecular mechanism by which high progesterone level prior to oocyte retrieval affect endometrial development is still unclear. We were particularly interested in the impact of high progesterone on the uNK cell count because the latter has been reported to be elevated in women with recurrent miscarriage and recurrent implantation failure. Does high progesterone level prior to oocyte retrieval have any impact on the morphological development and uNK cell count of the endometrium in the peri-implantation period (seven days after hCG administration) in women undergoing IVF treatment?Methods:This was a single-center, prospective cohort study carried out in a university-affiliated reproductive center between June2013and December2013. Endometrial biopsy was obtained from119subjects.All subjects included in the study were advised not to proceed with fresh embryo transfer in the treatment cycle but to have embryos frozen for replacement in a subsequent cycle, according to our establish clinical protocol to avoid ovarian hyperstimulation syndrome, either because there were more than15oocytes retrieved or because the progesterone level on the day of hCG administration was elevated to≥1.5ng/ml. Overall119women included in our study did not proceed to have fresh embryo transfer.In all the study cycles, luteal support continued until the day of endometrium biopsy, and consisted of intramuscular progesterone in oil at40mg/day for1day, starting from the night of oocyte retrieval and then at60mg/day for2days and then at80mg/day for3days.A total of119endometrial samples were collected using a Pipelle sampler (Prodimed, France) seven days after hCG administration (hCG+7) from the uterine fundus.All the specimens were assessed by two independent observes, experienced in the use of dating criteria of Noyes at al.(1950) to evaluate morphological development. The observers were blinded to the clinical details of the specimens. uNK cell count were performed by two independent observers. The inter-observer variability were assessed.All subjects had2consecutive blood tests for estrogen and progesterone.Stepwise multiple regression analysis was used to identify factors (progesterone level on the day of hCG administration, progesterone level after the day of hCG administration, estradiol level on the day of hCG administration, estradiol level after the day of hCG administration, duration of ovarian stimulation, and infertility years) affecting the results of histological dating or uNK cell count.According to a consecutive series of5828samples routinely collected from patients undergoing ovarian stimulation treatments for IVF from2011-2013in our hospital, the90%percentile was used as the cut-off to define high progesterone level, which was1.7ng/ml on the day of hCG administration and9.5ng/ml on the day after hCG administration.The results of histological dating, the proportion of disparity between glands and stroma, and the uNK cell number were further compared between subjects with normal progesterone and high progesterone. Results:Association of progesterone with endometrial development1. The results of stepwise multiple regression analysis showed that only the progesterone level after the day of hCG administration showed an association with histological dating.2. According to progesterone level on the day after hCG administration was normal or high (cut-off value:9.5ng/ml), overall119subjects were divided into two groups:the results of observer1:the endometrium was significantly advanced in women with high progesterone on both days (8.0±0.6days&7.6±0.7days,P<0.001); the results of observer2:the endometrium was also significantly advanced in women with high progesterone on both days (7.6±0.8days&6.8±1.0, P<0.001)3. The results of histological dating by the two observers were significantly (r=0.394, P<0.001) correlated to each other. Overall,70%of the results are within one day of each other, and95%of the results are within two days of each other.4. the proportion of glandular-stroma asynchrony defined as significant disparity between development of glandular and stroma component by4or more days, was significantly (P<0.001) higher in women with normal progesterone (51%) compared with women with high progesterone (22%). Association of progesterone with uNK cell number5. The results of stepwise multiple regression analysis showed that only the progesterone level on the day of hCG administration showed an association with the number of uNK cell.6. The results of the inter-observer variability of uNK cell count showed significant correlation (r=0.9, P<0.05) with a mean (±SD) of the absolute difference in uNK cell count between the observers of2.4%(±3.3%)7. According to progesterone level on the day of hCG administration was normal or high (cut-off value:1.7ng/ml), overall119subjects were divided into two groups:the median(range) of uNK cell count in women with high progesterone level (9.2%(1.3-21.6%))was significantly (P<0.001) higher than that of women with normal progesterone (6.3%(1.4-18.7%))Conclusion:1.High progesterone level prior to oocyte retrieval produced advancement in endometrial development as well as increased number of uNK cell count.2.2. The adverse effect of high progesterone prior to oocyte retrieval in IVF treatment cycle may be mediated via its action on the uNK cell. PART THREE:The effect of elevated progesterone levels before oocyte retrieval in women undergoing ovarian stimulation for IVF treatment on the genomic profile of peri-implantation endometriumAim:To investigate the impact of high progesterone level prior to oocyte retrieval on the genomic profile of endometrium in peri-implantation period, and in particular, the extent to which it affects the uNK cell pathwayMethods:This is a single-center, prospective cohort study. A total of12women undergoing IVF treatment who did not proceed to have fresh embryo transfer were included in our study. After oocyte retrieval, luteal support were given to all12subjects following our routine policy. A total of12endometrial samples were collected using a Pipelle sampler (Prodimed, France)7days after hCG administration from the uterine fundus.All subjects had blood sample taken for estrogen and progesterone measurement on the day of hCG and the day after hCG administration. According to a consecutive series of5828samples routinely collected from patients undergoing ovarian stimulation treatments for IVF from2011-2013in our hospital, the90%percentile was used as the cut-off to define high progesterone level, which was1.7ng/ml on the day of hCG administration and9.5ng/ml on the day after hCG administration.Four groups of patients were included in this study, depending on whether or not the progesterone level on the day of hCG administration and the day after hCG administration were elevated. Group1refers to subjects with normal progesterone level on both days (normal normal, NN); Group2refers to subjects with normal progesterone level on the day of hCG administration and high progesterone level on the day after hCG administration (normal high, NH); Group3refers to subjects with high progesterone level on the day of hCG administration and normal progesterone level on the day after hCG administration (high normal, HN); Group4refers to subjects with high progesterone level on both days (high high, HH). Three subjects were included in each group, with a total of12subjects included in this study.An absolute fold change of2.0or more and a corresponding corrected P-value<0.05were used to define the genes that had altered expression.Differential expressed genes between different groups were compared and selected in the following order:group4(HH) vs. group1(NN), group3(HN) vs. group1(NN), and group2(NH) vs. group1(NN). The lists of differential expressed genes were further submitted to a website based genomic functional analysis tool, DAVID. Pathway analysis between various groups were performed by using KEGG pathway provided by DAVIDUsing the list of differentially expressed genes found between Group4(HH) and Group1(NN), clustering analysis were perform for overall12samples.A total of8genes were selected for validation by qRT-PCR. Among these8genes,4genes were involved in natural killer cell mediated cytotoxicity pathway, including CD247molecular (Cd247), killer cell lectin-like receptor subfamily C, member2(KLRC2), killer cell lectin-like receptor subfamily D, member1(KLRD1), natural cytotoxicity triggering receptor1(NCR1), and another4genes were aquaporin-3(AQP3), leucine rich repeat containing1(LRRC1), matrix metalloproteinase (MMP26), and calcium-binding protein S100P. Results:1. The comparisons of the demographic features and outcome of ovarian stimulation in the treatment cycle of4groups of subjects showed no obvious differences between the groups2. The results of pathway analysis based on a total of153dysregulated genes (Group4compared to Group1) using DAVID showed that four major pathways have been affected, in descending order of the number of genes affected, natural killer cell mediated cytotoxicity (15), graft-versus-lost disease (6), antigen procession and presentation (6), and starch and sucrose metabolism (6).3. Using the same approach, the results of pathway analysis between Group3and Group1showed three major pathways have been affected, in descending order of the number of genes affected:cell cycle (8), spliceosome (7), natural killer cell mediated cytotoxicity (7).4. The results of pathway analysis between Group2and Group1showed that five major pathways have been affected, in descending order of the number of genes affected: cell cycle (7), spliceosome (7), natural killer cell mediated cytotoxicity (7),.wnt signaling pathway (7), and antigen procession and presentation (5).5. Natural killer cell mediated cytotoxicity pathway was consistently affected in all three groups of subject with high progesterone levels on at least one of these two days, compared with the remaining group of subject with normal progesterone on both days. In women with high progesterone level on both days, the natural killer cell mediated cytotoxicity pathway was the most prominent pathway affected, with a total of15genes dysregulated.6. The15altered genes involved in NK cell pathways (group4compared to group1) were KLRC2(up-regulated3.2folds), klrc3(up-regulated2.2folds), KLRD1(up-regulated3.4folds), KLRK1(up-regulated2.4folds), Ncrl (up-regulated3.1folds), GZMB (up-regulated2.4folds), HCST(up-regulated2.5folds), Fcgr3a (up-regulated2.3folds), Cd247(up-regulated2.3folds), FCER1G (up-regulated2.8folds), TYROBP(up-regulated2.6folds),Prfl (up-regulated2.4folds), rac2 (up-regulated2.3folds), Klrcl(up-regulated3.2folds), and TNFSF10(down-regulated2.4folds)7. Using153differentially expressed genes found between Group4(HH) and Group1(NN), the result of sample clustering for the overall12samples showed that each group did appear to have a distinctive genomic pattern. Among the four groups, Group2(Group NH) and Group3(Group HN) appeared to be closer to each other than the rest.8. The results of qRT-PCR validation for genes:CD247, KLRC2, KLRD1, NCR1, AQP3, LRRC1, MMP26, S100P were both consistent with the results of micro-array analysis.Conclusion:1. Progesterone level on the day of hCG administration and progesterone level on the day after hCG administration both significantly effect the endometrial gene expression profile.2. High progesterone prior to oocyte retrieval significantly altered the NK cell mediated natural cytotoxic pathway.